Amnesic shellfish poisoning was first recognized in 1987 at Prince Edward Island, Canada, after several people died and over one hundred became ill following the consumption of blue mussels (Bates et al., Canadian Journal of Fisheries and Aquatic Science 46:1203-1215 (1989); Perl et al., N. Eng. J. Med. 322:1775-1778 (1990)). The causative agent was identified as domoic acid, a naturally occurring neuroexcitatory amino acid produced by the pennate diatom Pseudonitzschia pungens (Grunow) Hasle forma multiseries (Hasle) Hasle (Subba Rao et al., Can. J. Fish Aquatic Sci 45:2076-2079 (1988); Bates et al., Canadian Journal of Fisheries and Aquatic Science 46:1203-1215 (1989); Douglas, et al., Can. J. Fish. Aquatic Sci. 49:85-90 (1992); Hasle, G. R., Nova Hedwigia, Beiheft 106:315-321 (1993)). Mussels that fed upon a bloom of this diatom concentrated the associated toxin, serving as a vector for human poisonings. Prior to this event, domoic acid was not considered a public health concern nor was it associated with blooms of diatoms. In following years, reports of toxic Pseudonitzschia blooms, as well as findings of domoic acid in shellfish, became more prevalent, but were restricted to eastern North America (Subba Rao et al., Can. J. Fish Aquatic Sci 45:2076-2079 (1988); Shumway, S. E., World Aquaculture 20:65-74 (1989); Martin et al., Mar. Ecol. Prog. Ser. 67:177-182 (1990). However, in 1991, the death of brown pelicans and cormorants in the vicinity of Santa Cruz, California (Monterey Bay), subsequent shellfish bans in Oregon and Washington, and a limited number of minor, human poisonings were also linked to this toxin, being the first recorded domoic acid outbreak in western North America (Anonymous, Communicable Diseases Summary, Oregon Health Division, Portland 40:1-2 (1991); Work et al., Toxin Marine Phytoplankton (Ed. by Graneli, et al.), pp. 643-649 (1993)). The pelican and cormorant mortalities coincided with a massive bloom of P. australis Frenguelli, the associated toxin of which was concentrated by feeding anchovies (Buck et al., Marine Ecological Progress Series 84:293-302 (1992); Fritz et al., Journal of Phycology 28:439-442 (1992); Garrison et al., Journal of Phycology 28:604-607 (1992); Work et al., Toxin Marine Phytoplankton (Ed. by Graneli, et al.), pp. 643-649 (1993)).
Domoic acid poisonings in eastern and western North America have drawn considerable attention to the potential for future outbreaks (Wood, et al. (eds.), Domoic Acid, Final Report of the Workshop, Oregon Institute of Marine Biology, Feb. 21-23, 1992 (1993)). In addition, there are concerns of the toxin's transfer through the food web, bio-accumulation and possible bio-transformation. The global distribution of Pseudonitzschia spp. shown to produce domoic acid also suggests the problem could be widespread (Hallegraeff, G. M., Phycologia 32:79-99 (1993); Villac et al., J. Shellish Res. 12:457-65 (1993); Villac et al., Hydrobiologia 267/270:213-24 (1993)). Not surprisingly, the study of Pseudonitzschia species has intensified, a trend that reflects both the public's and scientific communities' concerns over the safety of sea food and human's potential impact on the coastal environment.
Toxic Pseudonitzschia species can be difficult to identify, and in most cases require electron microscopy and an expert taxonomist for unambiguous classification. Consequently, detection and enumeration of such species in field samples is both challenging and labor intensive, especially when large numbers of samples are to be processed on a routine basis. In turn, "early warning" of potential domoic acid outbreaks may be compromised, particularly in areas where bloom cycles of Pseudonitzschia are poorly characterized, or in regions that harbor multiple, toxic and non-toxic Pseudonitzschia species, or when expert taxonomists are unable to immediately examine suspect samples.
In a step towards circumventing these problems, Douglas et al., Natural Toxins 2:166-174 (1994) and Scholin et al., Natural Toxins 2:152-165 (1994) showed that Pseudo-nitzschia species harbour unique ribosomal RNA sequences, suggesting a genetic basis from which one might delineate potentially toxic from non-toxic congeners. Ribosomal RNA (rRNA) sequences have been used since the late 1970s to define evolutionary and taxonomic relationships of numerous life forms (Woese et al., Proceedings of the National Academy of Science, U.S.A. 74:5088-5090 (1977); De Rijk et al., Nucleic Acids Research 22:3495-3501 (1994); Van de Peer et al. Nucleic Acids Research 22:3488-3494 (1994). Comparison of rRNA sequences from different organisms has shown that the molecule is comprised of a mosaic of conserved and variable domains. Ribosomal RNAs also occur at high copy number per cell, thus providing a naturally amplified hybridization target per cell (Vaheri et al. (eds.), Rapid Methods and Automation in Microbiology and Immunology, Springer-Verlag, NY (1991)).
In view of the potential contamination of sea food by toxigenic algal blooms, what is needed in the art is a means to rapidly detect and quantify Pseudo-nitzschia species from a marine sample. In particular, what is needed is an assay for single or multiple species of the genus Pseudo-nitzschia by detecting species-specific regions of rRNA accessible under non-denaturing conditions. The present invention provides these and other advantages.